| Abstract | We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE gels after boiling in Laemmli's sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts. |
| Authors | Naomi S Hachiya, Takuya Ohkubo, Yoshimichi Kozuka, Mineo Yamazaki, Osamu Mori, Hidehiro Mizusawa, Yuji Sakasegawa, Kiyotoshi Kaneko
(Affiliation: Second Department of Physiology, Tokyo Medical University, Shinjuku-ku, Japan.)
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| Journal | Analytical biochemistry
(Anal Biochem)
Vol. 347
Issue 1
Pg. 106-11
(Dec 1 2005)
ISSN: 0003-2697 United States |
| PMID | 16229812
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
|
| Chemical References |
- Saccharomyces cerevisiae Proteins
- L-Lactate Dehydrogenase
- DLD2 protein, S cerevisiae
|
| Topics |
- Aged
- Electrophoresis, Polyacrylamide Gel
- Female
- Humans
- Immunoblotting
(methods)
- Inclusion Bodies
(chemistry, immunology)
- L-Lactate Dehydrogenase
(chemistry)
- Lasers
- Male
- Microdissection
- Pick Disease of the Brain
(metabolism, pathology)
- Protein Denaturation
- Protein Folding
- Saccharomyces cerevisiae Proteins
(chemistry)
- Sensitivity and Specificity
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